Dyes for labelling molecular ligands

ABSTRACT

Cyanine dyes with improved fluorescence intensity and photostability.

BACKGROUND OF THE INVENTION

The present invention relates generally to fluorescent dye reagents.

Fluorescent dyes are widely used for labeling, detecting, andquantifying components in a sample. Analytical methods that utilize suchdye reagents include fluorescence microscopy, fluorescence immunoassay,flow cytometric analysis of cells, and various other applications. Thechoice of fluorescent dyes is particularly important in applicationsthat utilize multiplex, multicolor analysis.

Many fluorescent dyes currently used in aqueous systems have a tendencyto form dimers or aggregate compromising their performance. Moreover,many dyes in current use exhibit poor chemical and/or photochemicalstability. Thus, there is a continued need for the development of newfluorescent dye compounds. The present invention satisfies this need andprovides related benefits as well.

SUMMARY OF THE INVENTION

In some aspects, embodiments disclosed herein relate to a compound offormula I:

wherein

CI is a charge balancing counterion and p is an integer from 1-7;

m is 1, 3, 5, 7, or 9;

each incidence of X is independently hydrogen, halogen, alkyl- oraryloxy, alkyl- or arylthio, amino- or substituted amino, aryl or alkyl,any of which may be optionally substituted with a substituent selectedfrom carboxyl, sulfonate, sulfinate, sulfoxide, sulfone, sulfonamide,hydroxyl, amino, alkylamino, dialkylamino, alkoxy, and halogen;optionally, one or more X groups are combined to form a carbocyclicring;

Y is independently selected from O, NR_(a), S, Se, CR_(b)═CR_(c) andCR₂R₃;

Z is independently selected from O, NR_(a), S, Se, CR_(b)═CR_(c) andCR₄R₅;

-   -   wherein R_(a), R_(b), R_(c), R₂, R₃, R₄, and R₅ are        independently selected from alkyl, aryl or substituted alkyl        (such as aminoallyl), carboxyalkyl, sulfoalkyl or substituted        aryl (such as carboxyaryl or sulfoaryl);

R₁ is selected from aryl (for example phenyl, naphthyl), aralkyl oralkyl, any of which may be optionally substituted with at least onesubstituent selected from carboxyl, sulfonate, sulfinate, sulfoxide,sulfone, sulfonamide, hydroxyl, amino, alkyl(aryl)amino,dialkyl(aryl)amino, alkoxy, aryloxy, halogen, succinimidyl ester, ester,and amide, mono- or disubstituted amide;

R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈ areindependently selected from carboxyl, sulfonate, sulfinate, sulfoxide,sulfone, sulfonamide, hydroxyl, amino, alkylamino, dialkylamino, alkoxy,halogen, alkyl succinimidyl ester, ester, amide, or any ortho-disposedpair of R₆, R₇, R₈, R₉, R₁₉, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈is combined to form a further fused aromatic or heterocyclic ring whichis optionally substituted; wherein at least one of R₁₄, R₁₅, R₁₆, R₁₇,or R₁₈ is sulfonate or a further fused aromatic ring derived from R₁₄,R₁₅, R₁₆, R₁₇, or R₁₈ comprises at least one sulfonate substituent; withthe proviso that none of R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈ are carboxyl.

In other aspects, embodiments disclosed herein relate to a method ofmaking the aforementioned compounds of formula I. The method includescondensing a compound comprising formula IIA or IIB:

wherein Z═OR, SR, NHR or NR; w=0, 1, 2, 3

with a compound comprising formula IIIA or IIIB appropriately:

wherein Y₁ is NHR, NR_(d)R_(e), or XR_(o), (where X═O, S); w is 1, 2 or3.

To improve yield of unsymmetrical dye structure I from startingmaterials IIA and IIIB, synthesis may achieved by a two-step reactionwith preliminary formation of hemicyanines JIB and IIIA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph comparing the fluorescent intensities of exemplarydyes A and B with increasing dye/protein ratio.

FIG. 2 shows an overlay of the absorption spectra of Dye 3 of thepresent invention with commercial dye Cy3.

FIG. 3 shows a comparison of wavelength/fluorescence intensity of Dye 5Aand 5B of the present invention with commercial dye Dy682.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed, in part, to a fluorescent dyecompound of formula I:

wherein

CI is a charge balancing counterion and p is an integer from 1-7;

m is 1, 3, 5, 7, or 9;

each incidence of X is independently hydrogen, halogen, alkyl- oraryloxy, alkyl- or arylthio, amino- or substituted amino, aryl or alkyl,any of which may be optionally substituted with a substituent selectedfrom carboxyl, sulfonate, sulfinate, sulfoxide, sulfone, sulfonamide,hydroxyl, amino, alkylamino, dialkylamino, alkoxy, and halogen;optionally, one or more X groups are combined to form a cyclic ring;

Y is independently selected from O, NR_(a), S, Se, CR_(b)═CR_(c) andCR₂R₃;

Z is independently selected from O, NR_(a), S, Se, CR_(b)═CR_(c) andCR₄R₅;

Wherein R_(a), R_(b), R_(c), R₂, R₃, R₄, and R₅ are independentlyselected from alkyl, aryl or substituted alkyl like aminoallyl,carboxyalkyl, sulfoalkyl; substituted aryl like carboxyaryl, sulfoaryl

R₁ is selected from aryl (for example phenyl, naphthyl), or alkyl, anyof which may be optionally substituted with at least one substituentselected from carboxyl, sulfonate, sulfinate, sulfoxide, sulfone,sulfonamide, hydroxyl, amino, alkyl(aryl)amino, dialkylamino, alkoxy,aryloxy, halogen, succinimidyl ester, ester, and unsubstituted mono- ordisubstituted amide;

R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈ areindependently selected from carboxyl, sulfonate, sulfinate, sulfoxide,sulfone, sulfonamide, hydroxyl, amino, alkyl(aryl)amino, dialkylamino,alkoxy, aryloxy, halogen, alkyl succinimidyl ester, ester, amide, or anyortho-disposed pair of R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅,R₁₆, R₁₇, and R₁₈ is combined to form a further fused aromatic orheterocyclic ring which is optionally substituted; wherein at least oneof R₁₄, R₁₅, R₁₆, R₁₇, or R₁₈ is sulfonate or a further fused aromaticring derived from R₁₄, R₁₅, R₁₆, R₁₇, or R₁₈ comprises at least onesulfonate substituent; with the proviso that none of R₁₄, R₁₅, R₁₆, R₁₇,and R₁₈ are carboxyl.

Fluorescent dyes of the invention can be used as markers in manydifferent ways for the analysis of a major part of clinically,biologically, biochemically or chemically relevant substances, such asfor example cells, antibodies, proteins, hormones, nucleic acids,oligonucleotides, naturally occurring or modified nucleotides,carbohydrates and some others.

Fluorescent dyes are known to be particularly suitable for biologicalapplications in which highly sensitive detection is needed. Polymethinedyes derivatives often possess very narrow and intense absorption bands;their extinction coefficients and fluorescence quantum yields usuallyhigher than those of other types of dyes having comparable absorptionmaximum. Moreover, such derivatives possess high sensitivity to theirmolecular environment, have greater selective absorption and betterphotostability, which are important parameters, especially for singlemolecular analysis.

Some indodicyanine dyes exhibit strong fluorescence and highphotostability. However, their utility is limited when the cyanine dyesare employed in aqueous solution due, in part, to the tendency to formaggregates [Angew. Chem. 49 (1936) 563; Nature 138 (1936) 1009]. Thesepolymeric dye systems, in contrast with the individual molecule, exhibita markedly changed absorption and fluorescence behaviour. As a result ofthe aggregation, the absorption maximum may be shifted in comparisonwith the monomer dye molecule and more significantly they fluoresce muchmore poorly.

In some aspects, the present invention provides new cyanine dyes whichdo not exhibit the aforementioned drawbacks and which in particularexhibit a low tendency towards aggregation of the dyes themselves andare suitable for application as optical and especially fluorescentlabels in the visible or near infrared region. In some embodiments,polymethine dyes of the present invention, due to specific substituentplacement in dye molecule possess strong fluorescence, very low tendencyto form dimers or higher aggregates and possess very high photo- andchemical stability. Due to specific structure of new dyes, they exhibithigher fluorescence and selectivity of light absorption and increasedphoto- and chemical stability relative to dyes of the same structuralclass. Higher fluorescence and increased photo- and chemical stabilityof new dyes are demonstrated below in the Examples. Without being boundby theory, the N-aryl substituents are believed to provide the dyemolecule improved rigidity in terms of intramolecular vibronicinteractions; prevent from intermolecular interactions, includingreciprocal action with solvents, oxygen and other molecules, resultingin lower non-radiating energy loss from the excited state of such typeof cyanine dyes in solution.

Negatively charged dyes of the invention can be prepared with up toeight sulfonic groups in one dye molecule shown below:

Again, without being bound by theory, such increasing of negative chargebearing by the dye molecule dramatically decreases its tendency towardsdye-dye interaction and aggregation (especially when used in the contextof multi-dye constructs or in “poly-labelled” systems both in solutionas well as on the target especially “polymeric” molecules) and decreasedtendency towards non-specific interactions of the dye at an arbitrarybiomolecule (like DNA).

Sulfonic (or sulfonato) acid groups can be incorporated into indolium orbenzoindolium end groups via sulfoalkyl group attachment to nitrogenand/or C-3 carbon atoms of indocyanine dyes as exemplified by cyaninedyes A and B below (WO 2005/044923).

Moreover, as shown in FIG. 1, it has been indicated that negativelycharged dye A having two sulfonate groups is more readily self-quenchedwith increasing dye concentration relative to dye B with six sulfonicgroups.

In the present invention, hydrophilic negatively charged sulfonic-,sulfonato-groups are disposed on aromatic groups in compounds ofFormula 1. Without being bound by theory, the out-of-plane twist of theN-phenyl substituent and the presence of a solvent sphere “around” thenegatively charged sulfonate group act synergistically to decrease thetendency towards dye-dye interaction and aggregation resulting in theobserved fluorescence in water solution, as shown in the Examples below.The 1-phenyl substituent in heterocyclic dye 3H-indolium end-group(N-Aryl) is not planar relative to the rest of dye molecule and thusprovides a steric barrier to dye aggregation as well as aggregation withother non-dye molecules. This property can be useful, for example, inapplications that employ dyes in conjunction with nucleic acids orproteins, where dye-nucleic or dye-protein interactions can besignificant.

By contrast N-alkyl substituted dyes known in the art do not providesuch effect. Moreover from space-filling models of fragments of dyes offormula 1, one can observe that introducing a 1-phenyl substituent inheterocyclic dye 3H-indolium end groups (with an additional hydrophilicsulfonic group) increases the solvent surface around the molecule, whichis further expected to deteriorate dye-dye interaction and aggregationin water solution and also additionally decrease the tendency towardsnon-specific interactions of the dye at an arbitrary biomolecule (likeDNA) or surface. From a prototype dye based on N-methyl substitutedindolium derivatives (like commercial dyes) and based on new N-arylsubstituted indolium derivatives, one can observe how significantlydecreased ability of the dye molecule to aggregate from even oneend-group (N-phenyl substituted) compare to the other (N-methyl).

In some embodiments, dye compounds of the invention exhibit enhancedphotostability. It has been indicated that interaction of a dye moleculewith oxygen (O₂*) or per-hydroxy (OOH) radicals are the predominantpathways for dye photo-degradation. Identification of products of suchtype reactions have been demonstrated as shown in the scheme below:

It has been postulated that the photo-bleaching ratio depends on Red-Oxpotentials of dyes and correlate with availability of carbon atoms(especially those next to nitrogen atom) in a conjugation polymethinechain for interactions with active species. Thus, cyanine dyes of theinvention, due at least in part to the N-aryl groups not only benefitfrom decreased aggregation but improved photochemical stability as well.

In some embodiments, compounds of Formula 2 can be used as startingmaterials for dye synthesis.

Wherein: Z are −, or an appropriate organic or inorganic counterion; Xare —O—, —S—, —CR₇R₈; the dotted curve represents the non-metal (carbonor hetero-) atoms required to form one to three a benzo-, naphtho- orheterocyclic condensed rings having 5 to 7 atoms in each ring; R₃ and R₄groups are attached to the rings;

R is hydrogen atom, halogen atom, alkyl group, alkoxy group,unsubstituted or substituted amino group, carboxy-, sulfo-,alkylsulfono- or arylsulfono-, sulfonato-, unsubstituted or substitutedsulfonamido-, hydroxy-, unsubstituted or substituted (preferablysubstituted with hydrophilic or functional groups) alkyl/aryl groups;

R₃, R₄, each independently is hydrogen atom, halogen atom, alkyl group,alkoxy group, unsubstituted or substituted amino group, carboxy-,sulfo-, alkylsulfono- or arylsulfono-, sulfonato-, unsubstituted orsubstituted sulfonamido-, hydroxy-, unsubstituted or substituted(preferably substituted with hydrophilic or functional groups)alkyl/aryl groups;

R₇ and R₈ each independently represents alkyl, aryl or substitutedalkyl, aryl groups (preferably substituted with hydrophilic orfunctional groups alike sulfo-, sulfonato, sulfonamido-, carboxy-,hydroxy-, amino-alkyl/aryl groups) or they can form a part of cyclic, orheterocyclic groups; characterized in that at least one of R₃ and R₄groups is an amino-, substituted amino-carboxy-, carboxamido-, sulfo-,unsubstituted or substituted sulfonamido-, sulfonato-, alkylsulfono- orarylsulfono-, group or alkyl/aryl groups having substituents mentionedabove; R₁ represents unsubstituted or substituted aryl or heteroarylgroups preferably substituted with hydrophilic groups like sulfo- orsulfonato- groups.

Reagents of Formula 2 can be used in the preparation of dyes of thepresent invention such as those shown below:

and also for preparation of another type of cyanine dyes such as thefollowing:

as well as for the preparation of other different classes of polymethinedyes like styryl and merocyanine dyes:

Without being bound by theory, such dyes may have not only higherphoto-stability and strong fluorescence and possess larger Stocks shiftof maximum of fluorescence band. Stocks shift is a parameter forfluorescent labels and represents a distance between absorption andfluorescence maximums.

For the synthesis of dyes of the invention, the reaction of SO₃ or itscomplexes or their solutions in organic or inorganic solvents withsubstrates like quaternary heterocyclic salts formula (3.1) orappropriate methylene bases was applied.

Wherein: Z are −, or an appropriate organic or inorganic counterion; Xare —O—, —S—, —CR₇R₈; the dotted curve represents the non-metal (carbonor hetero-) atoms required to form one to three a benzo-, naphtho- orheterocyclic condensed rings having 5 to 7 atoms in each ring; R₃ and R₄groups are attached to the rings;

R₁ represents unsubstituted or substituted aryl or heteroaryl groups

For example, starting from quaternary heterocyclic salts formula 3.2

Wherein: Z are −, or an appropriate organic or inorganic counterion; Xand Y are independently selected from the group consisting of —O—, —S—,—CR₇R₈, each dotted curve independently represent the non-metal (carbonor hetero-) atoms required to form one to three a benzo-, naphtho- orheterocyclic condensed rings having 5 to 7 atoms in each ring; R₃, R₄,R₅ and R₆ groups are attached to the rings;

R₁, R₇ and R₈ each independently represents alkyl, aryl, heteroaryl orsubstituted alkyl, aryl, heteroaryl groups (preferably substituted withhydrophilic or functional groups alike, sulfonamido-, carboxy-,hydroxy-, amino-alkyl/aryl groups) or they can form a part of cyclic, orheterocyclic groups; R₃, R₄, each independently is hydrogen atom,halogen atom, alkyl group, alkoxy group, amino group, carboxy-,sulfono-, sulfonamido-, hydroxy-, unsubstituted or substituted(preferably substituted with hydrophilic or functional groups)alkyl/aryl groups.

In some embodiments, the present invention provides a method ofpreparing organic compounds (heterocyclic derivatives) having theformula (I-2) by interaction of SO₃ or its complexes or their solutionsin organic or inorganic solvents

with substrates like quaternary heterocyclic salts formula (3.1, 3.2)

The term “alkoxy,” as used herein refers to an alkyl ether group,wherein the term alkyl is as defined below. Examples of suitable alkylether groups include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,iso-butoxy, sec-butoxy, tert-butoxy, and the like.

The term “alkyl,” as used herein refers to a straight-chain orbranched-chain alkyl group containing from 1 to 20 carbon atoms. Incertain embodiments, the alkyl group will comprise from 1 to 12 carbonatoms. In further embodiments, the alkyl group will comprise from 1 to 6carbon atoms, which can optionally be used interchangeably with the term“lower alkyl” group. In yet further embodiments, the alkyl group willcomprise from 1 to 4 carbon atoms, which can also be usedinterchangeably with the term “lower alkyl” group. Alkyl groups can beoptionally substituted as defined herein. Examples of alkyl groupsinclude methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl and the like. The term “alkylene” as used hereinrefers to a saturated aliphatic group derived from a straight orbranched chain saturated hydrocarbon attached at two or more positions,such as methylene (—CH₂—). Unless otherwise specified, the term “alkyl”can include “alkylene” groups.

The term “aryl,” as used herein means a carbocyclic aromatic systemcontaining one, two or three rings wherein such polycyclic ring systemsare fused together. The term “aryl” include, without limitation,aromatic groups such as phenyl, naphthyl, anthracenyl, and phenanthryl.

The term “aralkyl” or “arylalkyl,” as used herein refers to an arylgroup, as defined herein, attached to the parent molecular moietythrough an alkyl group, as defined herein. An exemplary aralkyl is thebenzyl group. Other aralkyl groups include, without limitation,phenylethyl, phenylpropyl, phenylbutyl, naphthylmethyl, naphthylethyl,napthylpropyl, anthracenylmethyl, anthracenylethyl, phenanthrylmethyl,and phenanthrylethyl.

The term “aralkoxy” or “arylalkoxy,” as used herein refers to an arylgroup, as defined herein, attached to the parent molecular moietythrough an alkoxy group, as defined herein.

The term “alkene” or radical fragment “alkenyl,” as used herein refersto a straight-chain or branched-chain hydrocarbon group having one ormore double bonds and containing from 2 to 20 carbon atoms. In someembodiments, an alkene will comprise from 2 to 6 carbon atoms. The term“alkenylene” refers to a carbon-carbon double bond system attached attwo or more positions such as ethenylene [(—CH═CH—),(—C::C—)]. Examplesof suitable alkenyl groups include ethenyl, propenyl, 2-methylpropenyl,1,4-butadienyl and the like. The term “alkenyl” can include “alkenylene”groups.

The term “alkenyloxy,” as used herein refers to an alkenyl ether group,wherein the term alkenyl is defined herein. Examples of suitable alkenylether groups include allyloxy (2-propenoxy), vinyloxy (ethenoxy),1-propenoxy, n-butenoxy, and the like.

The term “alkyne” or radical fragment “alkynyl,” as used herein refersto a straight-chain or branched chain hydrocarbon group having one ormore triple bonds and containing from 2 to 20 carbon atoms. In certainembodiments, the alkynyl group comprises from 2 to 6 carbon atoms. Infurther embodiments, the alkynyl group comprises from 2 to 4 carbonatoms. The term “alkynylene” refers to a carbon-carbon triple bondattached at two positions, such as ethynylene (—C:::C—, —C≡C—). Examplesof alkynyl groups include ethynyl, propynyl, hydroxypropynyl,butyn-1-yl, butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, andthe like. The term alkynyl can include alkynylene groups.

The term “alkynyloxy” refers to an alkynyl ether group. Examples ofsuitable alkynyl ether groups include, ethynyloxy, 1-propynyloxy,propargyloxy (2-propynyloxy), butynyloxy, and the like.

The term “hydroxyalkyl” refers to an alkyl group bearing a hydroxylmoiety (—OH) on at least one carbon atom of the alkyl chain.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine, oriodine.

The term “bond,” as used herein refers to a covalent bond between twoatoms and can include single, double, and triple bonds.

The term “optionally substituted” means the anteceding group can besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group can include any of the substituentsdefined herein including, without limitation, one or more substituentsindependently selected from the following groups or a particulardesignated set of groups, alone or in combination: lower alkyl, loweralkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lowerheterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl,lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl,aryloxy, lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl,carboxyl, lower alkylcarbonyl, lower carboxyester, lower carboxamido,cyano, hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino,amido, nitro, thiol, lower alkylthio, lower haloalkylthio, lowerperhaloalkylthio, arylthio, sulfonate, sulfonic acid, trisubstitutedsilyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H, pyridinyl, thiophene,furanyl, lower carbamate, and lower urea. Two substituents can be joinedtogether to form a fused five-, six-, or seven-membered carbocyclic orheterocyclic ring consisting of zero to three heteroatoms, for exampleforming methylenedioxy or ethylenedioxy. An optionally substituted groupcan be unsubstituted (e.g., —CH₂CH₃), fully substituted (e.g., —CF₂CF₃),monosubstituted (e.g., —CH₂CH₂F) or substituted at a level anywherein-between fully substituted and monosubstituted (e.g., —CH₂CF₃). Wheresubstituents are recited without qualification as to substitution, bothsubstituted and unsubstituted forms are encompassed. Where a substituentis qualified as “substituted,” the substituted form is specificallyintended. Additionally, different sets of optional substituents to aparticular moiety can be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.” The following substituent definitions areprovided, which are within the scope of substituents embraced by theterm optionally substituted, in addition to those substituents alreadydefined above.

As used herein the term “lower alkyl ester” refers to a C₁-C₆ alkylchain ester of a carboxylic acid. In some embodiments, a “lower alkylester” refers to a C₁-C₄ alkyl chain ester of a carboxylic acid.Representative esters include methyl, ethyl, propyl, butyl, pentyl, andhexyl esters. Any of the forgoing esters can be optionally branched.Such branched esters include iso-propyl esters, sec-butyl esters,iso-butyl esters and tert-butyl esters, for example.

The term “alkylamino,” as used herein refers to an alkyl group attachedto the parent molecular moiety through an amino group. Suitablealkylamino groups can be mono- or dialkylated, forming groups such as,for example, N-methylamino, N-ethylamino, N,N-dimethylamino,N,N-ethylmethylamino and the like.

The term “alkylidene,” as used herein refers to an alkenyl group inwhich one carbon atom of the carbon-carbon double bond belongs to themoiety to which the alkenyl group is attached.

The term “alkylthio,” as used herein refers to an alkyl thioether (R—S—)group wherein the term alkyl is as defined above and wherein the sulfurcan be singly or doubly oxidized. Examples of suitable alkyl thioethergroups include methylthio, ethylthio, n-propylthio, isopropylthio,n-butylthio, iso-butylthio, sec-butylthio, tert-butylthio,methanesulfonyl, ethanesulfinyl, and the like.

The terms “amido” and “carbamoyl,” as used herein refer to an aminogroup as described below attached to the parent molecular moiety througha carbonyl group, or vice versa. The term “C-amido” as used hereinrefers to a —C(═O)—NR2 group with R as defined herein. The term“N-amido” as used herein refers to a RC(═O)NH— group, with R as definedherein. The term “acylamino” as used herein embraces an acyl groupattached to the parent moiety through an amino group. An example of an“acylamino” group is acetylamino (CH₃C(O)NH—).

The term “amino,” as used herein refers to —NRR′, wherein R and R′ areindependently selected from the group consisting of hydrogen, alkyl,acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl,any of which can themselves be optionally substituted. Additionally, Rand R′ can combine to form heterocycloalkyl, either of which can beoptionally substituted.

The term “arylalkenyl” or “aralkenyl” as used herein refers to an arylgroup attached to the parent molecular moiety through an alkenyl group.

The term “arylalkynyl” or “aralkynyl” as used herein refers to an arylgroup attached to the parent molecular moiety through an alkynyl group.

The term “arylalkanoyl” or “aralkanoyl” or “aroyl” as used herein refersto an acyl group derived from an aryl-substituted alkanecarboxylic acidsuch as benzoyl, naphthoyl, phenylacetyl, 3-phenylpropionyl(hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl,4-chlorohydrocinnamoyl, and the like.

The term aryloxy as used herein refers to an aryl group attached to theparent molecular moiety through an oxy.

The terms “benzo” and “benz” as used herein refer to the divalent groupC6H4=derived from benzene. Examples include benzothiophene andbenzimidazole.

The term “carbamate” as used herein refers to an ester of carbamic acid(—NHCOO—) which can be attached to the parent molecular moiety fromeither the nitrogen or acid end, and which can be optionally substitutedas defined herein.

The term “O-carbamyl” as used herein refers to a —OC(O)NRR′ group, withR and R′ as defined herein.

The term “N-carbamyl” as used herein refers to a ROC(O)NR′— group, withR and R′ as defined herein.

The term “carbonyl” as used herein, when alone includes formyl [—C(O)H]and in combination is a —C(O)— group.

The term “carboxyl” or “carboxy” as used herein, refers to —C(O)OH orthe corresponding “carboxylate” anion, such as is in a carboxylic acidsalt. An “O-carboxy” group refers to a RC(O)O— group, where R is asdefined herein. A “C-carboxy” group refers to a —C(O)OR groups where Ris as defined herein.

The term “cyano” as used herein refers to —CN.

The term “cycloalkyl” or “carbocycle” as used herein refers to asaturated or partially saturated monocyclic, bicyclic or tricyclic alkylgroup wherein each cyclic moiety contains from 3 to 12 carbon atom ringmembers and which can optionally be a benzo fused ring system which isoptionally substituted as defined herein. In certain embodiments, thecycloalkyl will comprise from 5 to 7 carbon atoms. Examples of suchcycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, tetrahydronaphthyl, indanyl, octahydronaphthyl,2,3-dihydro-1H-indenyl, adamantyl and the like. “Bicyclic” and“tricyclic” as used herein are intended to include both fused ringsystems, such as decahydronaphthalene, octahydronaphthalene as well asthe multicyclic (multicentered) saturated or partially unsaturated type.The latter type of isomer is exemplified in general bybicyclo[1,1,1]pentane, camphor, adamantane, and bicyclo[3,2,1]octane.

The term “ester,” as used herein refers to a carboxy group bridging twomoieties linked at carbon atoms.

The term “ether,” as used herein refers to an oxy group bridging twomoieties linked at carbon atoms.

The term “haloalkoxy,” as used herein refers to a haloalkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “haloalkyl,” as used herein refers to an alkyl group having themeaning as defined above wherein one or more hydrogens are replaced witha halogen. Specifically embraced are monohaloalkyl, dihaloalkyl andpolyhaloalkyl groups. A monohaloalkyl group, for one example, can havean iodo, bromo, chloro or fluoro atom within the group. Dihalo andpolyhaloalkyl groups can have two or more of the same halo atoms or acombination of different halo groups. Examples of haloalkyl groupsinclude fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl,dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl,difluorochloromethyl, dichlorofluoromethyl, difluoroethyl,difluoropropyl, dichloroethyl and dichloropropyl. “Haloalkylene” refersto a haloalkyl group attached at two or more positions. Examples includefluoromethylene (—CFH—), difluoromethylene (—CF₂—), chloromethylene(—CHCl—) and the like.

The term “heteroalkyl,” as used herein refers to a stable straight orbranched chain, or cyclic hydrocarbon group, or combinations thereof,fully saturated or containing from 1 to 3 degrees of unsaturation,consisting of the stated number of carbon atoms and from one to threeheteroatoms selected from the group consisting of O, N, and S, andwherein the nitrogen and sulfur atoms can optionally be oxidized and thenitrogen heteroatom can optionally be quaternized. The heteroatom(s) O,N and S can be placed at any interior position of the heteroalkyl group.Up to two heteroatoms can be consecutive, such as, for example,—CH₂—NH—OCH₃.

The term “heteroaryl,” as used herein refers to a 3 to 7 memberedunsaturated heteromonocyclic ring, or a fused monocyclic, bicyclic, ortricyclic ring system in which at least one of the fused rings isaromatic, which contains at least one atom selected from the groupconsisting of O, S, and N. In certain embodiments, the heteroaryl willcomprise from 5 to 7 carbon atoms. The term also embraces fusedpolycyclic groups wherein heterocyclic rings are fused with aryl rings,wherein heteroaryl rings are fused with other heteroaryl rings, whereinheteroaryl rings are fused with heterocycloalkyl rings, or whereinheteroaryl rings are fused with cycloalkyl rings. Examples of heteroarylgroups include pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, pyranyl, furyl, thienyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl,isothiazolyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl,quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, indazolyl,benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl,benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl,benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl,tetrazolopyridazinyl, tetrahydroisoquinolinyl, thienopyridinyl,furopyridinyl, pyrrolopyridinyl and the like. Exemplary tricyclicheterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl,dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein each refer to a saturated, partially unsaturated, or fullyunsaturated monocyclic, bicyclic, or tricyclic heterocyclic groupcontaining at least one heteroatom as a ring member, wherein each theheteroatom can be independently selected from the group consisting ofnitrogen, oxygen, and sulfur. In certain embodiments, theheterocycloalkyl will comprise from 1 to 4 heteroatoms as ring members.In further embodiments, the heterocycloalkyl will comprise from 1 to 2heteroatoms as ring members. In certain embodiments, theheterocycloalkyl will comprise from 3 to 8 ring members in each ring. Infurther embodiments, the heterocycloalkyl will comprise from 3 to 7 ringmembers in each ring. In yet further embodiments, the heterocycloalkylcan comprise from 5 to 6 ring members in each ring. “Heterocycloalkyl”and “heterocycle” are intended to include sulfones, sulfoxides, N-oxidesof tertiary nitrogen ring members, and carbocyclic fused and benzo fusedring systems; additionally, both terms also include systems where aheterocycle ring is fused to an aryl group, as defined herein, or anadditional heterocycle group. Examples of heterocycle groups includeaziridinyl, azetidinyl, 1,3-benzodioxolyl, dihydroisoindolyl,dihydroisoquinolinyl, dihydrocinnolinyl, dihydrobenzodioxinyl,dihydro[1,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl,dihy-dropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl,isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl,tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. Theheterocycle groups can be optionally substituted unless specificallyprohibited.

The term “hydrazinyl” as used herein refers to two amino groups joinedby a single bond, i.e., —N—N—.

The term “hydroxy,” as used herein refers to —OH.

The term “hydroxyalkyl,” as used herein refers to a hydroxy groupattached to the parent molecular moiety through an alkyl group.

The term “imine” or “imino,” as used herein refers to RN═.

The term “iminohydroxy,” as used herein refers to N(OH)C— and N—O—.

The term “isocyanato” refers to a —NCO group.

The term “isothiocyanato” refers to a —NCS group.

The term “lower,” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 to 6 carbonatoms, inclusive. In some embodiments, lower means containing from 1 to4 carbon atoms, inclusive.

The term “lower aryl,” as used herein means phenyl or naphthyl, whichcan be optionally substituted as provided.

The term “lower heteroalkyl,” as used herein refers to a stable straightor branched chain, or cyclic hydrocarbon group, or combinations thereof,fully saturated or containing from 1 to 3 degrees of unsaturation,consisting of one to six atoms in which one to three can be heteroatomsselected from the group consisting of O, N, and S, and the remainingatoms are carbon. The nitrogen and sulfur atoms can optionally beoxidized and the nitrogen heteroatom can optionally be quaternized. Theheteroatom(s) O, N and S can be placed at any interior or terminalposition of the heteroalkyl group. Up to two heteroatoms can beconsecutive, such as, for example, —CH₂—NH—OCH₃.

The term “lower heteroaryl,” as used herein means either 1) monocyclicheteroaryl comprising five or six ring members, of which between one andfour of the members can be heteroatoms selected from the groupconsisting of O, S, and N, or 2) bicyclic heteroaryl, wherein each ofthe fused rings comprises five or six ring members, comprising betweenthem one to four heteroatoms selected from the group consisting of O, S,and N.

The term “lower cycloalkyl,” as used herein means a monocycliccycloalkyl having between three and six ring members. Lower cycloalkylscan be unsaturated. Examples of lower cycloalkyl include cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl.

The term “lower heterocycloalkyl,” as used herein means a monocyclicheterocycloalkyl having between three and six ring members, of whichbetween one and four can be heteroatoms selected from the groupconsisting of O, S, and N. Examples of lower heterocycloalkyls includepyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl,and morpholinyl. Lower heterocycloalkyls can be unsaturated.

The term “lower amino,” as used herein refers to —NRR′, wherein R and R′are independently selected from the group consisting of hydrogen, loweralkyl, and lower heteroalkyl, any of which can be optionallysubstituted. Additionally, the R and R′ of a lower amino group cancombine to form a five- or six-membered heterocycloalkyl, either ofwhich can be optionally substituted.

The term “mercaptyl” as used herein refers to an RS— group, where R isas defined herein.

The term “nitro,” as used herein refers to —NO₂.

The terms “oxy” or “oxa,” as used herein refer to —O—.

The term “oxo,” as used herein refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein refers to an alkyl group whereall of the hydrogen atoms are replaced by halogen atoms.

The terms “sulfonate,” “sulfonic acid,” and “sulfonic,” as used hereinrefer to the —SO₃H group and its anion as the sulfonic acid is used insalt formation.

The term “sulfanyl,” as used herein refers to —S—.

The term “sulfinyl,” as used herein refers to —S(O)—.

The term “sulfonyl,” as used herein refers to —S(O)₂—.

The term “N-sulfonamido” refers to a RS(═O)₂NR′— group with R and R′ asdefined herein.

The term “S-sulfonamido” refers to a —S(═O)2NRR′, group, with R and R′as defined herein.

The terms “thia” and “thio,” as used herein refer to a —S— group or anether wherein the oxygen is replaced with sulfur. The oxidizedderivatives of the thio group, namely sulfinyl and sulfonyl, areincluded in the definition of thia and thio.

The term “thiol,” as used herein refers to an —SH group.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′ group, with R and R′ asdefined herein.

The term “O-thiocarbamyl” refers to a —OC(S)NRR′ group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

Any definition herein can be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

In some embodiments, the present invention provides a compound ofFormula I or its mesomer as shown below:

-   -   wherein    -   CI is a charge balancing counterion and p is an integer from        1-7;    -   m is 1, 3, 5, 7, or 9;    -   each incidence of X is independently hydrogen, halogen, alkyl-        or aryloxy, alkyl- or arylthio, amino- or substituted amino,        aryl, or alkyl, any of which may be optionally substituted with        a substituent selected from carboxyl, sulfonate, sulfinate,        sulfoxide, sulfone, sulfonamide, hydroxyl, amino, alkylamino,        dialkylamino, alkoxy, and halogen; optionally, one or more X        groups are combined to form a carbocyclic ring;    -   Y is independently selected from O, NR_(a), S, Se, CR_(b)═CR_(b)        and CR₂R₃;    -   Z is independently selected from O, NR_(a), S, Se, CR_(b)═CR_(b)        and CR₄R₅;        Wherein R_(a,b,c), R₂, R₃, R₄, and R₅ are independently selected        from alkyl, aryl or substituted alkyl like aminoalyl,        carboxyalkyl, sulfoalkyl; substituted aryl like carboxyaryl,        sulfoaryl

R₁ is selected from aryl (for example phenyl, naphthyl) or alkyl, any ofwhich may be optionally substituted with at least one substituentselected from carboxyl, sulfonate, sulfinate, sulfoxide, sulfone,sulfonamide, hydroxyl, amino, alkylamino, dialkylamino, alkoxy, halogen,succinimidyl ester, ester, and amide;

R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈ areindependently selected from carboxyl, sulfonate, sulfinate, sulfoxide,sulfone, sulfonamide, hydroxyl, amino, alkylamino, dialkylamino, alkoxy,halogen, alkyl succinimidyl ester, ester, amide, or any ortho-disposedpair of R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈is combined to form a further fused aromatic or heterocyclic ring whichis optionally substituted; wherein at least one of R₁₄, R₁₅, R₁₆, R₁₇,or R₁₈ is sulfonate or a further fused aromatic ring derived from R₁₄,R₁₅, R₁₆, R₁₇, or R₁₈ comprises at least one sulfonate substituent; withthe proviso that none of R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈ are carboxyl.

In some embodiments, the present invention provides compounds of thestructure IA or mesomer thereof:

In some embodiments, the present invention provides compounds of thestructure IB or mesomer thereof:

wherein i and j are, independently, 0, 1, or 2; k is 1 or 2; and A, B,and C are independently, optional further ring fusion.

In some embodiments, dyes of the present invention provides compounds ofthe structure IC or mesomer thereof:

wherein i and j are, independently, 0, 1, or 2; k is 1 or 2; and A, B,and C are independently, optional further ring fusion.

In some embodiments, the present invention provides compounds having thestructure ID or mesomer thereof:

In some embodiments, compounds of the present invention have thestructure ID1 or mesomers thereof:

wherein i and j are, independently, 0, 1, or 2; k is 1 or 2; and A, B,and C are independently, optional further benzene ring fusion.

In some embodiments, the present invention provides compounds of thestructure IE or mesomer thereof:

In some embodiments, the present invention provides compounds having thestructure IE1 or mesomer thereof.

wherein i and j are, independently, 0, 1, or 2; k is 1 or 2; and A, B,and C are independently, optional further ring fusion.

In some embodiments, compounds of the present invention are selectedfrom:

In some embodiments, the present invention provides a method of making acompound of formula I, as described above, the method includingcondensing a compound comprising formula IIA or IIB:

Wherein Z═OR, SR, NHR or NR; w=0, 1, 2, 3

with a compound comprising formula IIIA or IIIB respectively:

wherein Y1 is NHR, NR_(d)R_(e), XR_(o), (where X═O, S); w is 0, 1, 2 or3 In some embodiments, the present invention provides dyes havingstructure 1-P or mesomer thereof:

wherein m=2n+1; n=0, 1, 2, 3 but to attain maximum absorption at 532 nmpreferably: n=1; X₁-X_(m)═H or alkyl or substituted alkyl; aryl orsubstituted aryl; arylalkyl or Heteroarylalkyl; which together or with Rmay form a cyclic or heterocyclic ring; heteroatom oralkyl(aryl)substituted heteroatom; R¹, R², R³, R⁴═H, Alkyl orsubstituted alkyl; Aryl or substituted aryl; Arylalkyl orHeteroarylalkyl; R⁵-R¹¹═H; Alkyl or substituted alkyl; Aryl orsubstituted aryl; Arylalkyl or Heteroarylalkyl; any of R¹-R¹¹ maybe ormay contain functional groups such as SO₃, SO₃H, SO₂NR₁₂R₁₃, COO, COOH,COOR₁₄, CONR₁₅R₁₆; halide, Amino or substituted amino, nitro, carbonyl,azido; and one of R¹-R¹⁶ substituents or combinations thereof cancontain saturated and/or unsaturated Carbon-Carbon or Carbon-Heteroatomsingle, double or triple bonds such as two, three or more neighboringgroups from R⁵-R¹¹ may form a cyclic/heterocyclic ring(rings)

R¹-R¹⁶═(CH2)_(L)CCH; (CH2)_(L)N3; (CH2)_(L)CN where L=1-5

Dyes of structure 1-P can be prepared from heterocyclic derivatives 2-P,3-P using both known and new compounds as starting materials by reactionwith intermediates commonly used in cyanine dyes chemistry:

Some 2-methyloxazolo[4,5-b]pyridinium derivatives (Dyes & Pigments 2005v 66 pp 135-142; Dyes & Pigments 2007 v 75 pp 466-473) and substituted2,3,3-trimethyl-3H-indolium derivatives (3) (Waggoner, US(1993)5268486)were used previously for some other types dye synthesis.

To improve the yield of unsymmetrical dye structure 1-P, synthesis yieldcan be improved by performing a two-step reaction where hemicyanines 4-Por 5-P are produced first.

wherein A=Heteroatom (N, O, S); R¹⁷, R¹⁸H, Alkyl, Aryl or Heteroaryl,COR¹⁹

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I 2,3,3-Trimethyl-1-(3-sulfophenyl)-3H-indolium-5-sulfonate

This Example shows the preparation of coupling partner CP-1, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 1 g (3 mmol) of 2,3,3-trimethyl-1-phenyl-3H-indoliumhydrosulphate and 3 ml fuming sulphuric acid was heated five hours at70° C. The product was precipitated with diethyl ether and washed withacetone and ethanol to provide 0.75 g (63%) of CP-1.

Example II 2,3,3-Trimethyl-1-phenyl-3H-indolium-5-sulfonate

This Example shows the preparation of coupling partner CP-2, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 1 g (4.25 mmol) of2-methylene-3,3-trimethyl-1-phenyl)-2,3-dihydro-1-H-indole and 1 mlfuming sulphuric acid was stirred at room temperature one hour thenheated at 70° C. for three hours. The product was precipitated withdiethyl ether and washed with acetone and ethanol to provide 0.7 g (52%)of CP-2.

Example III 3-Ethyl-2-methyl-benzothiazolium-6-sulfonate

This Example shows the preparation of coupling partner CP-3, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 0.5 g (4.25 mmol) of 2-methyl-3-ethyl-benzothiazoliumtosylate and 2 ml fuming sulphuric acid was stirred at room temperaturefor one hour then heated at 50° C. for three hours. The product wasprecipitated with diethyl ether and washed with acetone and ethanol toprovide 0.35 g (95.3%) of CP-3.

Example IV3-(3-Sulfopropyl)-2-methyl-5-methoxy-benzothiazolium-6-sulfonate

This example shows the preparation of coupling partner CP-4, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 0.3 g (1 mmol) of 2-methyl-5-metoxy-3-(3-sulfonatopropyl)benzothiazolium and 1 ml fuming sulphuric acid was stirred at roomtemperature for one hour then heated at 50° C. for three hours. Theproduct was precipitated with diethyl ether and washed with acetone andethanol to provide 0.25 g (66%) CP-4.

Example V 1-(3-Sulfopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate

This example shows the preparation of coupling partner CP-5, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 0.5 g (1.8 mmol) of1-(3-sulfonatopropyl)-2,3,3-trimethyl-3H-indolium and 2 ml fumingsulphuric acid was stirred at room temperature for one hour then heatedat 40° C. for three hours. The product was precipitated with diethylether and washed with acetone and ethanol to provide 0.7 g (99%) ofCP-5.

Example VI 3-(3-Sulfopropyl)-2-methyl-benzothiazolium-6-sulfonate

This Example shows the preparation of coupling partner CP-6, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 0.25 g (0.9 mmol) of3-(3-sulfonatopropyl)-2-methyl-benzothiazolium and 2 ml fuming sulphuricacid was stirred at room temperature one hour then heated at 25° C. for24 hours. The product was precipitated with diethyl ether and washedwith acetone and ethanol to provide 0.1 g (30%) of CP-6.

Example VII 1-Ethyl-2,3,3-trimethyl-3H-indolium-5-sulfonate

This Example shows the preparation of coupling partner CP-7, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 0.5 g (1.9 mmol) of 1-ethyl-2,3,3-trimethyl-3H-indoliumtosylate and 2.5 ml fuming sulphuric acid was stirred at roomtemperature for 24 hours. The product was precipitated with diethylether and washed with acetone and ethanol to provide 0.35 g (68%) ofCP-7.

Example VIII1-(5-Carboxypenthyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate

This Example shows the preparation of coupling partner CP-8, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 0.35 g (1 mmol) of1-(carboxypenthyl)-2,3,3-trimethyl-3H-indolium bromide and 2 ml fumingsulphuric acid was stirred at room temperature for 24 hours. The productwas precipitated with diethyl ether and washed with acetone and ethanolto provide 0.15 g (43%) of CP-8.

Example IX3-Ethyl-1,1,2-trimethyl-7-sulfo-1H-benzo[e]indolium-5-sulfonate

This Example shows the preparation of coupling partner CP-9, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 3.17 g (10 mmol) of3-ethyl-1,1,2,-trimethyl-1H-benzo[e]indolium-7-sulphonate and 10 mlfuming sulphuric acid was stirred at 25° C. for 48 hours then heated at70° C. for 5 hours. The product was precipitated with diethyl ether andwashed with acetone and ethanol to provide 3.5 g (88%) of CP-9.

Although sulfonated benzo[e]indolium systems have been incorporated intocyanine dyes, such compounds, exemplified by betaine CP-9A and6,8-disulfonated derivative CP-9B, are either only monosulfonated orpossess two sulfonate groups on the same single ring.

CP-9 and related dyes based on the 5,7-disulfonated benzoindoliumskeleton can exhibit improved photostability, provide improved watersolubility and can exhibit a diminished tendency to aggregate comparedto cyanine dyes based on their counterparts CP-9A and CP-9B. Moreover,the generality of the synthetic procedure is amendable to thepreparation of N-aryl-substituted analogues of quaternary benzoindoliumderivatives such as CP-9C below:

Example X 3-Ethyl-2-methyl-7-sulfo-naphtho[2,1-d]thiazolium-5-sulfonate

This Example shows the preparation of coupling partner CP-10, anintermediate useful in the preparation of dyes of the present invention.

A mixture of 0.5 g (1.25 mmol) of3-ethyl-2-methyl-naphtho[2,1-d]thiazolium tosylate and 5 ml fumingsulphuric acid was stirred at 70° C. for 5 hours. The product wasprecipitated with diethyl ether and washed with acetone and ethanol toprovide 0.3 g (62%) of CP-10, which could be further purified byconventional column chromatography.

Example XI 3-Ethyl-2-methyl-benzoxazolium-5- and3-ethyl-2-methyl-benzoxazolium-6-sulfonate

This Example shows the preparation of coupling partners CP-11 and CP12,intermediates useful in the preparation of dyes of the presentinvention.

A mixture of 0.33 g (1 mmol) of 2-methyl-3-ethyl-benzoxazolium tosylateand 2 ml fuming sulphuric acid was stirred at room temperature for onehour then heated at 50° C. for four hours. The product was precipitatedwith diethyl ether washed with acetone and ethanol to provide 0.1 g(41%) of a mixture of CP-11 and CP-12. The isomers are separated bycrystallisation from methanol with isomer CP-12 as the main fractionCP-11 isolated from the filtrate.

It has been indicated that cyanine dyes based on the benzoxazoliumstructure exhibit strong fluorescence and can exhibit improved stabilityrelative to indocarbo- and dicarbocyanine-based systems. Thus, dyesbased on coupling partners CP-11 and CP-12, as well as N-arylatedversions thereof, are expected to be strong fluorescent dye candidates.

Example XII

This Example shows the preparation of exemplary compound Dye 1 andcomparison of its physical properties.

A mixture of 80 mg (0.2 mmol) of2,3,3-trimethyl-1-(3-sulfophenyl)-3H-indolium-5-sulfonate (CP-1), 20 mg2-(4-carboxyphenyl)malonicdialdehyde, 0.5 ml pyridine in 1 ml of aceticanhydride was heated for three minutes at 145° C. under microwaveirradiation. The product was precipitated with diethyl ether andconverted to its sodium salt. The product was purified by HPLC providing0.02 g (25%) of Dye 1, which shows an absorption maximum at 658 nm inaqueous solution.

In a comparison of photo-stability of solutions of Dye 1 and Alexa 647in water, dye 1 shows improved photostability relative to thesecommercial dyes. After 25 days of irradiation the solution of Dye 1still possessed 85% of its initial fluorescence intensity.

Example XIII

This Example shows the preparation of exemplary compound Dye 2 and itsphysical properties.

A mixture of 0.395 g (1 mmol) of2,3,3-trimethyl-1-(3-sulfophenyl)-3H-indolium-5-sulfonate (CP-1, ExampleI) and 0.525 g (1 mmol)2-(4-acetanilidobutadienyl)-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolium-5-sulfonate:

in 3 ml of acetic anhydride and 0.3 g triethylamine was heated for 20minutes at 110° C. The acetic anhydride was removed under reducedpressure and the product precipitated with ethylacetate to provide 0.6 g(55%) of Dye 2, which could be further purified by HPLC.

Dye 2 exhibits an absorption maximum at 647 nm in water. Dye 2 stabilitywas also compared against the stability of Cy5 (GE Healthcare) in waterafter six days storage of a solution on a bench top. Dye 2 maintainedbetter fluorescence intensity over the six day test period. Dye 2stability was also measured by absorbance decay, with a comparison toCy5 and Alexa647 in water after irradiation by 660 nm laser. Dye 2 wasmore stable in water compared than the structurally similar commercialdyes Cy5 and Alexa647.

Example XIV

This Example shows the preparation of exemplary compound Dye 3 and itsphysical properties.

A mixture of 0.395 g (1 mmol) of2,3,3-trimethyl-1-(3-sulfophenyl)-3H-indolium-5-sulfonate (CP-1, ExampleI), 0.457 g (1 mmol)2-(2-anilinovinyl)-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolium-5-sulfonate:

in 5 ml of acetic anhydride and 0.3 g triethylamine was heated for 20minutes at 110° C. The acetic anhydride was removed under reducedpressure and the product precipitated with ethylacetate to provide 0.65g (68%) Dye 3, which could be further purified by HPLC. Dye 3 exhibitsan absorption maximum at 550 nm in water.

The fluorescence intensity of Dye 3 (331 a.u. at 575 nm) compared withCy3 (422 a.u. at 565 nm) in water at the same optical density ofsolution under excitation at 545 nm. FIG. 2 shows an overlay of theabsorption spectrum of Dye 3 with Cy3 in water. As shown in FIG. 2, thefluorescence max for the Dye 3 Red shifted with a slightly larger Stokesshift (about 25 nm) and as a result provides better separation of thefluorescence signal from the excitation wavelength. Moreover, theintegrated fluorescence intensity is higher for Dye 3 even at Cy3absorption maximum. Dye 3 also exhibits better stability over timecompared to Cy3, where after 14 days, the optical density of Cy3 is 85%compared to 94% with Dye 3.

Example XV

This Example shows the preparation of exemplary compound Dye 4 and itsphysical properties.

A mixture of 36 mg of1,1,3-trimethyl-3-(3-sulfophenyl)-1H-benzo[e]indolium-disulfonate(CP-13B):

and 10 mg malonic aldehyde dianil hydrochloride, 0.5 mL acetic anhydrideand 0.5 ml pyridine was heated at 120° C. for 5 minutes. The aceticanhydride removed under reduced pressure and the product precipitatedwith ethylacetate to provide 15 mg of Dye 4, which exhibited anabsorption maximum at 683 nm in water.

Dye 4 exhibited similar absorption as commercially available dyes forthe same spectral region: Dy681 and Dy682 (Dyomics GmbH, Jena, Germany).However, the fluorescence maximum for Dye 4 has bathochromic shift andas a result provides better separation of the fluorescent signal fromexcitation wavelength and less cross-talk with 660 dye. Dye 4 also hadhigher fluorescence intensity than Dy681 and Dy682.

Preparation of coupling partner 13B,1,1,3-Trimethyl-3-(3-sulfophenyl)-1H-benzo[e]indolium-disulfonatetriethylammonium salt 1,1,2-Trimethyl-3-phenyl-1H-benzo[e]indoliumperchlorate

N-Phenyl-N-(2-naphthyl)hydrazine (2.3 g) (or hydrochloride (2.7 g)) wasdissolved in 50 ml of ethanol and 10 g of 3-methyl-butanone-2 was added.To this mixture 10 ml 70% perchloric acid was added slowly with stirringat room temperature. Stirring was continued for one hour at 80-90° C.and after cooling down, the resultant precipitate was filtered off andwashed with ethanol to provide 0.6 g (16%)1,1,2-trimethyl-3-phenyl-1H-benzo[e]indolium perchlorate as a yellowsolid, which was sufficiently pure for the next synthetic step. ¹H NMR(400 MHz, TFA) δ 8.05 (d, 1H, J=8.5), 7.86 (t, 2H, J=7.6), 7.62 (dd, 4H,J=6.8, 13.4), 7.52 (t, 1H, J=7.6), 7.37 (d, 2H, J=7.3), 7.00 (d, 1H,J=8.9), 2.58 (s, 3H), 1.81 (s, 6H).

1,1-Dimethyl-2-methylen-3-phenyl-2,3-dihydro-1H-benzo[e]indole

1,1,2-Trimethyl-3-phenyl-1H-benzo[e]indolium perchlorate (11) (0.386 g)was suspended in 50 ml of benzene or diethyl ether and a solution 0.1 gpotassium hydroxide in 2 ml water was added. The mixture was stirred atroom temperature under nitrogen for one hour. The organic solvent layerwas separated and washed with water and filtered through silica gel andthen dried and evaporated. The resultant oily product (0.2 g) wassufficiently pure for the next synthetic step. ¹H NMR (400 MHz, DMSO) δ8.05 (d, 1H, J=8.5), 7.81 (d, 1H, J=8.1), 7.67 (d, 1H, J=8.8), 7.59 (t,2H, J=7.8), 7.43 (dt, 4H, J=6.9, 20.3), 7.22 (t, 1H, J=7.3), 6.75 (d,1H, J=8.7), 4.05 (d, 1H, J=1.7), 3.88 (d, 1H, J=1.7), 1.71 (s, 6H).

1,1,2-Trimethyl-3-phenyl-1H-benzo[e]indolium p-toluenesulfonate

1,1,2-Trimethyl-3-phenyl-1H-benzo[e]indolium perchlorate (3.86 g) wassuspended in 50 ml of diethyl ether and a solution of 0.5 g sodiumhydroxide in 5 ml water was added. The mixture was stirred at roomtemperature under nitrogen for half an hour. The ether layer wasseparated and washed with water and filtered through a thin layer ofsilica gel. To this solution 2 g of p-toluenesulfonic acid was added andthe mixture stirred for an hour. The pink product was filtered off andwashed with diethyl ether to provide 3.5 g (76%)1,1,2-Trimethyl-3-phenyl-1H-benzo[e]indolium p-tolyenesulfonate, whichwas of sufficient purity to carry on to the next synthetic step. ¹H NMR(400 MHz, TFA) δ 7.99 (d, 1H, J=8.5), 7.82 (m, 2H), 7.59 (m, 7H), 7.48(t, 1H, J=7.6), 7.29 (m, 2H), 7.05 (t, 3H, J=9.2), 6.95 (d, 1H, J=9.0),2.52 (s, 3H), 2.14 (s, 4H), 1.75 (s, 6H)

Sulfonated 1,1,2-Trimethyl-3-phenyl-1H-benzo[e]indolium

Fuming sulphuric acid (30%, 3 ml) was added at temperature below 0° C.to 1,1-dimethyl-2-methylen-3-phenyl-2,3-dihydro-1H-benzo[e]indole (0.57g, 2 mmol) or 1,1,2-trimethyl-3-phenyl-1H-benzo[e]indoliump-tolyenesulfonate (0.9 g, 2 mmol) with cooling and stirred for 2 hours.The mixture was kept at room temperature for two hours and then heatedat 80° C. for 12 hours. The solution was carefully diluted with drydiethyl ether and the oily product separated and washed with ether.Isomeric coupling partners CP-13A and CP-13B were separated by HPLC.Both compounds have strong fluorescence in water: CP-13A—green,CP-13B—blue.

Example XVI

This Example shows the preparation of exemplary compound Dye 5A and Dye5B and their physical properties.

A mixture of appropriate1,1,3-trimethyl-3-(3-sulfophenyl)-1H-benzo[e]indolium-disulfonatefraction (CP-13A or B), and 2-(4-carboxyphenyl)malonic aldehyde in amixture of acetic anhydride and pyridine was heated at 120° C. for 15minutes. The acetic anhydride was removed under reduced pressure and theproduct precipitated with ethylacetate.

Alternatively, a mixture of appropriate1,1,3-trimethyl-3-(3-sulfophenyl)-1H-benzo[e]indolium-disulfonatefraction (CP-13A or B), and 2-(4-carboxyphenyl)malonic aldehyde dianilhydrochloride:

in a mixture of acetic anhydride and pyridine was heated at 120° C. for5 min. The acetic anhydride was removed under reduced pressure. Theproduct precipitated with ethylacetate and filtered off.

Dye 5A, from CP-13A, has an absorption maximum at 686 nm in water andDye 5B, from CP-13B, has an absorption maximum at 688 nm. A comparisonof Dye 5A and 5B with commercially available Dy681 and Dy682 shows thatthe fluorescence intensity for Dye 5A and 5B are higher than thecommercial dyes. Moreover, the fluorescence maximum for Dye 5A and 5Bare red shifted and as a result providing better separation of thefluorescent signal from the excitation wavelength and less cross-talkwith 660 dye. Finally, Dyes 5A and 5B are brighter than Dy681 and Dy681and exhibit better stability as shown in FIG. 3.

Example XVII

This Example shows the preparation of exemplary compound Dye 6 and itsphysical properties.

A mixture of appropriate1,1,3-trimethyl-3-ethyl-7-sulfo-1H-benzo[e]indolium-5-sulfonate (CP-9,Example IX) and 2-(4-carboxyphenyl)malonic dialdehyde in aceticanhydride was heated at 140° C. for 5 min. The acetic anhydride wasremoved under reduced pressure and the product precipitated withethylacetate.

Alternatively, for N-arylated analogues, a mixture of appropriate1,1,3-trimethyl-3-(3-sulfophenyl)-1H-benzo[e]indolium-disulfonatefraction CP-13A or CP-13B, and 2-(4-carboxyphenyl)malonic aldehydedianil hydrochloride in mixture of acetic anhydride and pyridine washeated at 120° C. for 5 min. Acetic anhydride removed under reducedpressure then product precipitated with ethylacetate and filtered off.

Dye 6 exhibited an absorption maximum at 673 nm in water. Dye 6 shows ahigher relative fluorescence intensity.

Example XVIII

This Example shows the preparation of exemplary compound Dye 7 and itsphysical properties.

A mixture of 0.395 g (1 mmol) of2,3,3-trimethyl-1-(3-sulfophenyl)-3H-indolium-5-sulfonate (CP-1, ExampleI) and 0.455 g (1 mmol)2-(4-anilinobutadienyl)-3,3-dimethyl-5-carboxy-1-(3-sulfonatopropyl)-3H-indolium:

in 3 ml of acetic anhydride and 0.3 g triethylamine was heated for 20minutes at 110° C. The acetic anhydride was removed under reducedpressure and the product precipitated with ethylacetate to provide 0.6 g(55%) of Dye7 which was purified by HPLC. Absorption max 650 nm (water).NR5-125 Abs. Max 650 nm

Example XIX

A mixture of 1 mmol appropriate1,1,3-trimethyl-3-(3-sulfophenyl)-1H-benzo[e]indolium-disulfonatefraction B (5B), and 0.525 g (1 mmol)2-(4-acetanilidobytadienyl)-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolium-5-sulfonate(3),

3 ml of acetic anhydride and 0.3 g triethylamine was heated 20 minutesat 110° C. Acetic anhydride removed under reduced pressure then productprecipitated with ethylacetate. Yield 0.6 g (55%). Product was purifiedby HPLC. Absorption max 664 nm (water). Fluorescence max 687 nm (water).This new dye (NR665) has more than 3 times stronger fluorescence inwater compare with Dy681

Example XX

A mixture of 1 mmol appropriate1,1,3-trimethyl-3-(3-sulfophenyl)-1H-benzo[e]indolium-disulfonatefraction B (5B), and 0.525 g (1 mmol)2-(4-acetanilidobytadienyl)-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolium-5-sulfonate(3),

3 ml of acetic anhydride and 0.3 g triethylamine was heated 20 minutesat 110° C. Acetic anhydride removed under reduced pressure then productprecipitated with ethylacetate. Yield 45%. Product was purified byflash-column. Absorption max 567 nm (water).

A few examples of symmetrical N-phenyl-substituted indocyanine dyes areknown such as dyes 1, 2

see for example: EP 1134613 (2001); US 2005/0013966, JP 2002226731.These dyes have fluorescence properties and therefore they found somepractical application: WO 96/10620; US 2004/6835431. However, such dyescan have insufficient fluorescence in water or biological systems. Tocompare fluorescent properties of dyes based on unknown beforesulfonated N-arylindole derivatives and known prototype (2) some new dyewith one (NR 647 S1) and t(NR 647 S2) N-phenyl-indole moiety wereprepared.

Spectral properties for solutions of new symmetrical dyes in waterpresented in Table 1.

TABLE 1 Absorption Fluorescence Fluorescence Dye R₁ R₂ max, nm max, nmintensity, % S0 (2) H H 649 674 100 S1 SO₃ ⁻ H 653 676 170 S2 SO₃ ⁻ SO₃⁻ 653 676 190

The family of indocyanine dyes based on new startingmaterials—sulfonated N-aryl-indole derivatives have significantlystronger fluorescence (up to two times).

Further investigations reveal that solutions of new dyes (such as NR647S2 and NR647 S1) not only have stronger fluorescence but they are morephoto-stable than prototype (2) as well. One of the modern applicationsof cyanine dyes based on their fluorescent properties is chemicallylabelling of different bio-molecules. For such applications a dyemolecule can be modified with a functional group like amino orcarboxy-groups, which making them suitable for coupling. For thispurpose a series of unsymmetrical dyes with one functional carboxy- andat least one sulfonic group have been prepared.

Table 2 shows the fluorescence properties of unsymmetricalN-arylindodicarbocyanines in water.

TABLE 2 Absorption Fluorescence Fluorescence Dye R1 R2 max, nm max, nmintensity, % NR 647-0 H H 648 670 100 NR 647-1 SO3− H 649 672 125 NR647-2 SO3− SO3− 648 670 120

This family of unsymmetrical indocyanine dyes based on new startingmaterials—sulfonated N-aryl-indole derivatives have quite strongfluorescence and they can be useful for different applications, whichare using fluorescent dyes chemically conjugated to differentbio-molecules. The present invention provides dyes that have improvedfluorescent properties compared to various commercial dyes such as Cy3,Cy5, and Alexa647 for the same spectral region

Example XXI

This Example shows the preparation of Dye 9.

Dye structure 9 has been prepared in accordance with embodimentsdisclosed herein. Some details provided below

This Example shows the preparation of exemplary compound Dye 9 and itsphysical properties. A mixture of 1.34 g (10 mmol)2-methyl-oxazolo[4,5-b]pyridine and 2 ml ethyliodide in a closed systemwas kept 2 h at 110° C. The product was precipitated with acetone andfiltered off. The yellow crystalline product was filtered off and washedwith diethyl ether to provide 2.47 g (86%)4-ethyl-2-methyl-oxazolo[4,5-b]pyridinium iodide, which was ofsufficient purity to carry on to the next synthetic step.

A mixture of 0.29 g (1 mmol) 4-ethyl-2-methyl-oxazolo[4,5-b]pyridiniumiodide and 0.2 ml ethylisoformalilide was kept 1 h at 100° C. Theproduct was precipitated with ether and filtered off. The crystallineproduct was filtered off and washed with diethyl ether to provide 0.3 g(76%) 4-ethyl-2-(2-phenylamino)vinyl-oxazolo[4,5-b]pyridinium iodide,which was of sufficient purity to carry on to the next step of dyesynthesis.

Dye 9. Method A

A mixture of 0.29 g (1 mmol) of4-ethyl-2-methyl-oxazolo[4,5-b]pyridinium iodide, 0.457 g (1 mmol)2-(2-anilinovinyl)-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolium-5-sulfonate:

3 ml of acetic anhydride and 0.3 g triethylamine were heated 20 minutesat 110° C., acetic anhydride-removed under reduced pressure then theproduct precipitated with diethyl ether. The product was purified byHPLC. The absorption maximum of equimolar solutions in ScanMix of newdye 9 was 536 nm, 0.162 a.u.

Dye 9. Method B

A mixture of 0.39 g (1 mmol) of4-ethyl-2-(2-anilino)vinyl-oxazolo[4,5-b]pyridinium iodide, 0.35 g (1mmol) 2,3,3-trimethyl-1-(5-carboxypentyl)-3H-indolium-5-sulfonate, 3 mlof acetic anhydride and 0.13 g of sodium acetate was heated 20 minutesat 110° C. Acetic anhydride was removed under reduced pressure then theproduct precipitated with diethyl ether. The product was purified byflash column. Spectral properties investigation of dye 9 revealed thatthis representative of unsymmetrical indocyanine dyes has an absorptionmaximum at about 536 nm closer to the laser excitation wavelength (532nm). This new dye 9 (max 583 nm, 447 a.u.) has stronger fluorescencecompared with DG527 (594 nm, 401 a.u.).

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1-11. (canceled)
 12. A compound or a mesomer thereof, wherein thecompound is:


13. The compound of claim 12, which is

or a mesomer thereof.
 14. The compound of claim 12, which is

or a mesomer thereof.
 15. The compound of claim 12, which is

or a mesomer thereof.
 16. The compound of claim 12, which is

or a mesomer thereof.
 17. The compound of claim 12, which is

or a mesomer thereof.
 18. A compound or a mesomer thereof, wherein thecompound is: